Gene therapy is a process by which genes are introduced into cells which then become mini-factories which manufacture and release essential compounds in cells and tissue which improve the life of the patient. Gene therapy has the potential to revolutionize the treatment of genetic disorders, diseases associated with a genetic component like cancer, AIDS, and many other diseases. Gene therapy may be the only remedy for some individuals who would otherwise die or be severely disabled. Gene transfer may also be employed for systemic protein and peptide-like hormone administration. Nucleic acid sequences coding for a protein (insulin, growth hormone) would be administered to the patient allowing endogenous production of their own medication.
Successful gene therapy requires the identification of an appropriate therapeutic gene for treatment of the disease, in addition to a delivery system by which that gene can be delivered to desired cell type both efficiently and accurately. Early attempts of gene transfer involved the removal of cells from the individual, and the alteration of the cells in the culture by the introduction of a functioning copy of the gene. The next step included grafting the genetically engineered cells back into the patient. This ex vivo approach to gene therapy is obviously limited to those target tissues that are not undergoing frequent multiplication and cell generation that could cause progressing elimination of the grafted cells. The ability of the altered cells to efficiently recombine with the target tissue is another limiting factor of the ex vivo approach since many cells do not exhibit the ability to recombine.
The limitation and the complexity of the ex-vivo approach facilitated the development of direct in vivo gene transfer methods. Direct gene therapy involves the administration of the gene into the body, targeting of the gene to the desired cells and into the nucleus of said genes, and expression of functioning gene products therein. Currently there are two different approaches for direct gene transfer. One is a viral approach and the other is a non-viral approach. Viral and non-viral gene therapies differ in the methods used to deliver genes to the target cells and direct the uptake of gene into the nucleus. Viral gene therapies employ genetically engineered viral particles to deliver the gene to target cell, and non-viral gene therapies employ gene delivery systems comprised of synthetic or semi-synthetic gene formulations. The limitations of viral therapies relate to the residual viral elements within the viral vectors which can be immunogenic, cytopathic, or recombinogenic.
Antisense technology has introduced the possibility of down-regulating or specifically turning off the expression of individual genes. This technology has enormous therapeutic potential. Antisense oligodeoxynucleotides (AON or ODN) constitute sequences of 15 to 21 nucleotides with the order of the nucleotides providing the molecule with the specificity to target genetic material. An oligonucleotide whose bases are tailored to complement part of a particular mRNA, can bind to and complex with that section of the mRNA. This can prevent gene expression which may prevent protein synthesis by passive or reactive inhibition of mRNA translation. Antisense ODN's to DNA seem to inhibit DNA transcription by formation of a triple helix.
Antisense oligonucleotides enter cells by pinocytosis and/or receptor-mediated endocytosis after binding to cell surface antigens. Uncharged oligomers enter cells by passive diffusion and charged oligomers enter by endocytosis. It seems that oligomers are not internalized by cells very efficiently. Methods for improving cellular uptake and biological efficacy of ODN's have been devised, including their conjugation to a synthetic polypeptide poly(L-lysine) tail with or without transferrin, or encapsulation in cationic or antibody targeted liposomes.
As with other modes of contemporary gene therapy, delivery remains a central and crucial issue. For example, Antisense oligonucleotides per se are thought not to cross the intact Blood Brain Barrier (BBB). There are no studies analyzing the passage of antisense oligomers across the Blood Brain Barrier. Attempts to deliver them across the BBB by hyperosmotic BBB disruption after conjugation or by incorporation into liposomes have, as a whole, been unsuccessful. Direct injection of free antisense results in their rapid breakdown.
Although, most research in vivo gene therapy has focused on the use of recombinant virus vectors, progress has been made toward developing non-viral formulations of gene for in vivo human gene therapy. The advantages of non-viral vectors are that they can introduce DNA into non-dividing cells, do not integrate into the chromosome, do not posses infective risk, and are potentially less expansive than viral vectors. The principle underlying non-viral gene delivery is that the problem of delivering DNA in vivo is not significantly different from the problem of delivering conventional drugs or biological products to intracellular compartment in the body. Non-viral gene therapies involve known drug delivery methods for the administration and targeting of genes to selected cells in vivo, where they express therapeutic products.
Various methods have been described for non-viral gene therapy, ranging from the direct administration of “naked” plasmid DNA to the systemic administration of complex synthetic formulations. Some approaches are aimed at developing “artificial viruses” that attempt to mimic the process of viral infection using synthetic or semi-synthetic components. Others apply the theory and method of advanced, particulate drug delivery to administer DNA to selected somatic targets. These approaches employ plasmid DNA complexes containing lipids, proteins, peptides, or polymeric carriers. The principle disadvantage associated with non-viral systems has been insufficient levels of gene expression, irreproducibility and significant variations in gene expression on various cell types.
The two classes of synthetic gene delivery systems that have been investigated most actively involve the use of either cationic liposomes or polycationic polymers. The assembly of these systems is achieved by an electrostatic condensation of the “anionic” DNA with the “cationic” moiety of either a lipid or a synthetic polymer. The cationic polymer-based systems have been most widely associated with the formulation of receptor-mediated gene delivery systems. This technique employs the ability of receptors on the surface of a variety of different cells to efficiently bind and internalize a ligand. Several ligands have been exploited for the efficient internalization of DNA-ligands complexes. These include: asialoorosmucoid and other galactosylated proteins which target the hepatic asialoglycoprotein receptor; transferrin which binds to the transferrin receptor and mannosyl which is recognized by the mannose receptor of macrophages. Targeting ligands are covalently linked to a polycation polymer, typically to poly(lysine) derivatives, and then form a ligand-poly(lysine)-DNA complex by the ionic interaction between the positively charged poly(lysine) and the negatively charged DNA. Often, an endosomolytic agent is added to the transfection mixture to induce endosomal lysis and enhance DNA release from the endosome in order to achieve high transfection efficiency. The efficiency of poly(lysine)-DNA conjugates in transfecting numerous cell types in vitro has been demonstrated, but their potential usefulness for in vivo human gene therapy is limited due to their cytotoxicity.
More advanced polymeric gene delivery systems employ macromolecules with a very high cationic charge density that act as an endosomal buffering system, thus suppressing the endosomal enzymes activity and protecting the DNA from degradation. The high cationic charge density mediates both DNA condensing and buffering capacity, that diminish the requirement for an endosomolytic agent addition.
Polymers Used in Gene Transfer
The polycations used for gene complexation are polyamines that become cationic at physiologic conditions. All polymers contain either primary, secondary, tertiary or quaternary amino groups capable of forming electrostatic complexes with DNA under physiologic conditions. The highest transfection activity is obtained at a 1.1–1.5 ratio of polycation to DNA. The most studied polyamines for gene transfer includes, poly(lysine) and its derivatives, polyamidoamine starburst dendrimers, polyethyleneimine, natural and modified polysaccharides, and acrylic cationic polymers. The details for each polymer class are described in Domb et al. (A. Domb, M. Levy, Polymers in gene therapy, Frontiers in Biological Polymer Applications, R. M. Ottenbrite (ed), Technomic, Vol. 2, 1999,1–16.).
Polycations may be more versatile for use than the liposomes and other conventionally used spherical gene carriers. Several polycations have been reported to induce gene expression for example diethylaminoethyl dextran and other cationized polysaccharides [F. D. Ledley, Huiman Gene Therapy, 6, 1129, 1995; Yamaoka et al. Chemistry Letters, 1171–72, 1998]. These polymers have little structural similarity with each other except possessing cationic groups.
Cationic polysaccharides have been used for gene delivery. Chitosan, a linear cationic polysaccharide was suggested by several authors for gene delivery [K. W. Leong et al, DNA-Chitosan nanospheres: Transfection efficiency and cellular uptake, Proceed. Intl. Symp. Control. Rel. Bioact. Mater. 24:75–76, 651–652, 671–674, 1997; R. Richardson, H. V. J. Kolbe, R. Buncan, Evaluation of highly purified chitosan as a potential gene delivery vector, Proceed. Intl. Symp. Control. Rel. Bioact. Mater. 24:649–650, 1997] DNA-chitosan nanospheres were found to be significantly less toxic than poly(L-lysine) or Lipofectin using the MTT test. Compared to standard Lipofectamine mediated gene transfer, these nanospheres yield lower levels of gene expression in HEK 293 (human embryonic kidney cells), IB3 (bronchial epithelial cells) and HTE (human tracheal epithelial cells). Surface modification of DNA/chitosan complex nanoparticles by covalently binding poly(ethylene glycol), transferrin and mannose-6-phosphate receptor to facilitate entry into cells and improve storage stability was also studied. The Purified and hydrophobized chitosan has also been suggested as carrier for genes [K. Y. Lee, I. C. Kwon, Y. H. Kim, W. H. Jo, S. Y. Jeong, Selfaggregates of hydrophobically modified chitosan for DNA delivery, Proceed. Intl. Symp. Control. Rel. Bioact. Mater. 24:651–652,1997].
Midox (WO 95/30020) describes a polypeptide such as polylysine modified at the g-amino group with a molecule bearing hydroxyl groups. Genzyme describes in WO 97/46223 lipid derivatives of short chain alkylamines such as spermine and spermidine for use in gene transfection. For example, one or two spermine or spermidine groups attached to cholesterol via an amide or carbamate bonds. WO 98/27209 to Emory Univ. describes a range of modified cationic polypeptides based on lysine for use in gene transfection.
The polymers described in the prior art can be grouped into two catagories: One including linear or dendrimeric polymers with random distribution of amino groups which are part of the polymer backbone such as poly(ethylene imines), poly(amido-amine) dendrimers, and poly(alkylamino-glucaramide). The second including linear polymers with a single primary secondary or tertiary amino group attached to the polymer units. Examples of such polymers are: poly(dimethylaminoethyl methacrylates), dimethylamino dextran, and polylysines.
All of the above polymers are polycations with a random distribution of the cationic sites. This randomness is probably the reason for the fact that these polymers may work for some nucleotides and cell types and not for others. Most of these polymers are toxic to cells and non-biodegradable, while the polymers based on amino acids such as polylysines are immunogenic.
It can be said that in the prior art, little attention was given to:    1. the structure of the polycation, the charge density and space distribution of cationic groups in the polymer to optimize complexation with anionic nucleotides;    2. the type of cationic groups, primary, secondary or tertiary groups were considered as cationic sites.
3. the toxicity and immunogenicity of the polymer;    4. the biodegradability and elimination properties of the polymer carrier;
In general, it has been believed that the cationic charge of the polymers is the main factor important for complexation and transfection. Also, these cationic polymers did not result in high enough transfection yield for commercial interest in ex-vivo experiments, in addition to animal experimentation. The degradation and elimination of the polymer carrier was not carefully treated and most polycations described for use in gene therapy are not biodegradable and/or toxic.
In designing a universal polycation system for gene delivery one should consider the way in which a plasmid becomes active in the cell and tissue. The plasmid has first to be protected from DNA degrading enzymes in the extracellular medium, then penetrate the cell wall, protected from degrading systems, i.e. the lisosome and enzymes, in the intracellular medium until it is internalized in the nucleus, penetrate into the nucleus and being released in its active form from the polymer carrier.
This invention describes a versatile and universal polycation system based on oligoamine grafted on natural or synthetic polysaccharides that is capable of complexing various plasmids and antisense, administering them into various cells in high yields and into the nucleus in active form to produce the desired protein.
It is the objective of the present invention to provide polycations that:    1. better fit the complexation requirements for effective delivery of a plasmid or an antisense;    2. biodegrade into non-toxic fragments at a controlled rate;    3. non-toxic and no-immunogenic in vivo;    4. form a stable enough complex with low and high molecular weight polynucleotides including therapeutic plasmids and antisense.    5. provide effective polymeric delivery system that result in a high transfection yield in a range of cells and in tissues.    6. can be reproducibly prepared at an affordable cost.
Another objective of this invention is to provide a controlled release of DNA in tissue or cell by complexing DNA with designed polymers that gradually de-complex and release the DNA or by incorporation of the complexed polynucleotides in a biodegradable matrix which will release the DNA in the site of insertion for periods of weeks and months.
Thus, according to the present invention there is provided a biodegradable polycation composition associated with an anionic macromolecule, said macromolecule being selected from the group consisting of a plasmid, an oligonucleotide, an antisense, a peptide, a protein, an anionic polysacharide and combinations thereof, comprising:                a) a polysaccharide chain having an amount of saccharide units ranging from 2 to 2000; and        b) at least one grafted oligoamine per 5 saccharide units, wherein said oligoamine is selected from the group consisting of a linear, branched and cyclic alkyl amine having at least two amino groups and said oligoamine has a molecular weight of up to 2000 daltons.        
In another preferred embodiment of the present invention said polysaccharide chain is selected from the group consisting of dextrans, arabinogalactan, pullulan, cellulose, cellobios, inulin, chitosan, alginates and hyaluronic acid.
In a further preferred embodiment of the present invention said saccharide units are connected by a bond selected from the group consisting of acetal, hemiacetal, ketal, orthoester, amide, ester, carbonate and carbamate
In an even further preferred embodiment of the present invention said polysaccharide is a synthetic polysaccharide formed from the condensation of an aldaric acid and a diaminoalkane.
In a preferred embodiment of the present invention said grafted oligoamine is grafted to said polysaccharide chain by a bond selected from the group consisting of amine, amide and carbamate. In another preferred embodiment the oligoamine has the formula:NH2—[CH2)x—N—(R)—CH2)y—N—(R′)—(CH2)z]—n—NH2 wherein x, y, z are an integer between 0 and 4 and x+y+z is between 1 and 4 and n is at least 1 when x+y+z=2 or more, or at least 2 when x+y+z=1 and wherein R and R′ groups are H or an aliphatic side group of 1 to 6 carbons.
In a further preferred embodiment of the present invention said oligoamine is selected from the group consisting of spermine and derivatives thereof.
In an even further preferred embodiment of the present invention said oligoamine is selected from the group consisting of a linear and branched ethyleneimine oligomer having up to 10 ethylene imine units.
In an even further preferred embodiment of the present invention said oligoamine is selected from the group consisting of a a peptide consisting of up to 20 amino acids with at least 50% contain a cationic side group including, lysine, ornithine, and diphthamic acid.
In a preferred embodiment of the present invention said amphiphilic residue is selected from the group consisting of fatty chains, phospholipids, cholesterol derivatives, ethylene glycol oligomers and propylene glycol oligomers, wherein said ethylene and propylene glycol oligomers have a fatty chain block on one side.
In a further preferred embodiment of the present invention said amphiphilic residue is connected to said polysaccharide chain by a bond selected from the group consisting of an amine, amide, imine, ester, ether, urea, carbamate and carbonate.
In an even further preferred embodiment of the present invention said amphiphilic residue facilitates the crossing of the polycation through biological membranes.
In a preferred embodiment of the present invention said polycation composition is not toxic or immunogenic.
In an even further preferred embodiment, the composition of the invention further comprises a ligand for facilitating the binding of said composition to a predetermined type of cell or tissue.
It is a further an objective of the invention to provide a pharmaceutical composition comprising the composition described above, in combination with a pharmaceutically acceptable carrier.
It is a further an objective of the invention to provide a pharmaceutical composition comprising the composition described above, in combination with amphiphilic cationic and/or nonionic lipids and cationic and nonionic polymers generally used for nucleotide delivery transfection. Examples of lipids include DOTMA, DOTAP, DMRIE, GAP-DLRIE, DODHF, aklylated spermine, and other derivatives described in: G. Byk and D. Scherman, Exp. Opin. Ther. (1998) 8(9): 1125–1141; D. A. Treco and R. F. Selden, non viral gene therapy, Molec. Med. Today, 1995, 1(7): 299–348).
The present invention describes a range of biodegradable polycations based on grafted oligoamine residues on synthetic or a natural polysaccharides which are effective in delivering plasmids and antisense for a high biological effect. The grafting concept where side chain oligomers are attached to either a linear or branched hydrophilic polysaccharide backbone, allows two/three dimensional interaction with an anionic surface area typical to the double or single strand DNA chain. This type of flexible cationic area coverage is not available with non-grafted polycations or low molecular weight cations. Low molecular weight amines and their lipid derivatives such as the lipofectin and lipofectamine have a localized effect on the DNA which the degree of complexation is dependent on how these small molecules organized around the anionic DNA. Each molecule has to be synchronized with the other molecules at all times of the transferction process whereas when the oligoamines are grafted on a polymer they are already synchronized and each side chain helps the other side chains to be arranged to fit the anionic surface of the given DNA. By grafting the functional groups is an average distribution along a polymer chain at a certain distance between each other (for example, grafting an oligoamine chain every one, two, three or four polymer unit may provide optimal complexation with various DNAs.
U.S. Pat. No. 4,146,515 (D4) describes oxidation of starch and reacting it in bulk with epichlorhydrin-dimethylamine or ammonia to improve the industrial properties of starch. The product is cationic but is not a grafted oligoamine conjugated to a polysaccharide.
WO93/25239 (D3) to Advanced Magnetics describes a range of derivatives of arabinogalactran. In Example 10 page 19, there is described the treatment of AG with galactose oxiddase (GO). The reaction details are given including the purification process and determination of the number of aldehyde groups formed. The oxidation with the enzyme takes place at room temperature which yields 0.34 milliequivalents of aldehydes. This is an alternative method of oxidation of AG and is not related to an oligoamine conjugated to polysaccharides.
In example 6, page 17 of said publication, there is described the reaction between polylysine and arabinogalactan (AG) at a ratio of 500 mg polylysine and 100 mg of AG in the present of cyanoborohydride as reducing agent. The resulting product yield was 30 mg which represents a 5% yield. No date is given on the analysis of this product besides a comment that the product contains an amine and saccharide and has a molecular weight of 25,000. Under these reaction conditions in which AG was not oxidized and native AG was used, the chance for a chemical conjugation between polylysine and AG is very low as there are no or only a few aldehyde groups on AG that are available for a reductive amination reaction. Indeed, the negligible yield of 5% with no characterized product indicates that probably nothing was obtained in this reaction and the 5% material isolated was either polylysine contaminated with AG or AG contaminated with polylysine, the latter seeming more probable as native AG has a molecular weight of 25,000. Thus this example does not teach or suggest to a person skilled in the art the formation of an oligoamine conjugated with polysaccharides.
WO 98 01162 (D1) describes the formation of Chitosan nanoospheres containing DNA complexed with the cationic groups of the native Chitosan. Page 17 and FIGS. 7 and 8 describe in detail the formation of the Chitosan nanospheres loaded with the DNA. Chitosan contains one amino group per saccharide unit as it is a polymer of glucose amine. This polymer is a polysaccharide with one amino side group which is not an oligoamine conjugated to a polysaccharide.
RU 2,027,190 (D2) describes a conjugate of polysaccaharide antigen and polyethylene imine as an immunosorbent for detecting streptococcal and pneumococcal infections. Said reference thus teaches an antigen which is chemically bound to a polysaccharide via a spacer which is a polyethylene imine and does not teach or suggest an oligoamine conjugated to a polysaccharide.
U.S. Pat. No. 5,567,685 describes the conjugation of various oxidizable drugs to a polysaccharide, however, does not teach or suggest an oligoamine conjugated to polysaccharide.
The use of biodegradable cationic polyol carriers is especially suitable for transfection and biological applications because they are water soluble and miscible in aqueous vehicles. The resultant grafted polymers are water soluble or dispersible in water, it can be readily transported to cells in vivo by known biological processes, and acts as an effective vehicle for transporting agents complexed with it.
The compositions of the present invention are composed of a natural or synthetic polysaccharide backbone with a grafted complexation functionality, i.e. aliphatic organic cationic residues containing at least two amino groups. The alkyl amino cationic residues are distributed in an optimal charge distribution tailored for as many plasmid or oligonucleotide for optimal transfection results. The polymer has hydrophobic/hydrophilic side groups that allow penetration of the polymer-plasmid complex into cells for transfection.